Millions of people in the U.S. are at risk for heart disease and resulting cardiac events. CVD and underlying atherosclerosis is the leading cause of death among all demographic groups, despite the availability of therapies directed at its multiple risk factors. Atherosclerosis is a disease of the arteries and is responsible for coronary heart disease associated with many deaths in industrialized countries. Several risk factors for coronary heart disease have now been identified: dyslipidemias, hypertension, diabetes, smoking, poor diet, inactivity and stress. The most clinically relevant and common dyslipidemias are characterized by an increase in beta-lipoproteins (very low density lipoprotein (VLDL) and LDL) with hypercholesterolemia in the absence or presence of hypertriglyceridemia (Fredrickson et al., 1967, N Engl J Med. 276:34-42, 94-103, 148-156, 215-225, and 273-281). There is a long-felt significant unmet need with respect to CVD with 60-70% of cardiovascular events, heart attacks and strokes occurring despite the treatment with statins (the current standard of care in atherosclerosis). Moreover, new guidelines suggest that even lower LDL levels should be achieved in order to protect high risk patients from premature CVD [National Cholesterol Education Program (NCEP), 2004].
PCSK9, also known as NARC-1, was identified as a protein with a genetic mutation in some forms of familial hypercholesterolemia. PCSK9 is synthesized as a zymogen that undergoes autocatalytic processing at the motif LVFAQ in the endoplasmic reticulum. Population studies have shown that some PCSK9 mutations are “gain-of-function” and are found in individuals with autosomal dominant hypercholesterolemia, while other “loss-of-function” (LOF) mutations are linked with reduced plasma cholesterol. Morbidity and mortality studies in this group clearly demonstrated that reducing PCSK9 function significantly diminished the risk of cardiovascular disease.
Of significant importance to the treatment of CVD, a LOF mutation may sensitize humans to statins, allowing for efficacy at a lower dose (hence, improving risks associated with safety and tolerance) and potentially achieving lower plasma cholesterol levels than with current therapies.
PCSK9 is secreted into the plasma predominantly by hepatocytes. Genetic modulation of PCSK9 in mice confirmed the ability of PCSK9 to regulate blood lipids, and suggested that it acts to down-regulate hepatic LDLR protein levels.
The mechanism by which, and the site at which, PCSK9 down-regulates LDLR protein has not been clearly established. When over-expressed, PCSK9 may act both within the hepatocyte and as a secreted ligand for LDLR. There is strong evidence that extracellular PCSK9 binds to cell surface LDLR and promotes LDLR degradation at an intracellular site. However, it is also possible that PCSK9 could interact with the LDLR when the two proteins are translated within the endoplasmic reticulum (ER) and traffic through endosomal compartments towards the cell membrane. Maxwell et al., 2005, Curr. Opin. Lipidol. 16:167-172, showed that PCSK9-mediated LDLR endocytosis and degradation was not altered by proteosome inhibitors nor was it modulated by different classes of lysosomal and nonlysosomal proteases. Two naturally occurring familial hypercholesterolemia mutations, S127R and D129G, have been reported to be defective in autoprocessing and secretion as levels of these mutant proteins were greatly reduced or undetectable in the media of transfected cells. Yet these mutants demonstrated an enhanced ability to down-regulate LDLR, consistent with their identification in individuals with high plasma LDL (Homer et al., 2008, Atherosclerosis 196:659-666; Cameron et al., 2006 Human Molecular Genetics 15:1551-1558; Lambert et al., 2006, TRENDS in Endocrinology and Metabolism 17:79-81. Since these mutants apparently do not get secreted extracellularly, and yet do downregulate LDLR, this strongly suggests that an intracellular site of action is physiologically important.
From the information available in the art, and prior to the present invention, it remained unclear whether the introduction of an antibody-, peptide-, or aptamer-based PCSK9 antagonist into the blood circulation to selectively antagonize extracellular PCSK9 would be effective to reduce hypercholesterolemia and the associated incidence of CVD and, if so, what properties of a PCSK9 antagonist are needed for such in vivo effectiveness.